IDENTIFICATION OF 2 ESCHERICHIA-COLI PSEUDOURIDINE SYNTHASES THAT SHOW MULTISITE SPECIFICITY FOR 23S RNA

Several putative Escherichia coli pseudouridine (Psi) synthases have b een identified by iterative searching of genomic databases for ORFs ho mologous to known Psi synthases [Gustafsson et al. (1996) Nucleic Acid s Res. 24, 3756-3762]. Of these, yceC and yfil were proposed to encode Psi synthases which modify 23S rRNA. In the present work, yceC and yf il were cloned and overexpressed in E. coli, and the encoded enzymes, YceC and Yfil, were purified to homogeneity. Both proteins converted U rd residues of rRNA to Psi, thus confirming their identities as Psi sy nthases. However, in in vitro experiments both enzymes extensively mod ified Urd residues of both 23S rRNA and 16S rRNA. Gene-disruption of y ceC resulted in the absence of Psi modification at positions U955, 250 4,and 2580 of 23S RNA, thus identifying these sites as in vivo targets for YceC. Likewise, yfil disruption resulted in the absence of Psi mo dification at positions U1911, 1917, and possibly 1915 of 23S RNA. Dis ruption of yceC did not affect the growth under the conditions tested, whereas yfil-disrupted cells showed a dramatic decrease in growth rat e. Since YceC and Yfil hypermodify RNA in vitro, factors in addition t o ribonucleotide sequence must contribute to the in vivo specificity o f these enzymes.